Field of the Invention
The present invention is in the field of methods and compositions for amplification, including methods and compositions for isothermal whole genome amplification (WGA) of single cells.
Description of the Related Art
Standard isothermal WGA protocols instruct technicians to use at least 10 ng (or 1,500 cell equivalents) of starting genomic material to carry out a successful isothermal WGA reaction. Groups have tried and failed to perform single cell isothermal WGA. For example, standard procedures for isothermal WGA on single cell genomic material have resulted in undesirable outcomes, such as high allele dropout (ADO) and locus dropout (LDO) rates, with some reports of >30% for ADO using ≥10 ng of starting genomic material (Morrison et al., Am J Trop Med Hyg. 76 (2007) 1132-1137). Standard procedures have also demonstrated amplification bias and “noise” in array comparative genomic hybridization (array CGH) output. Further, standard cell lysis conditions performed prior to WGA often result in damage (e.g., nicking, breaking, and/or fragmenting) of the genetic material, which can compromise the performance of subsequent methods such as WGA and array CGH (Kumar et al., Biotechniques 44 (2008) 879-890). Because of these problems, the preferred method for WGA from a single cell has been a non-isothermal, or PCR-based, method.
Further, isolating genomic DNA from whole blood requires extensive purification. Standard protocols, such as that provided with the GENOMIPHI™ DNA Amplification Kit (GE Healthcare, Waukesha, Wis.), indicate that blood components such a heme can inhibit Phi29 DNA polymerase. Studies in which these protocols have been used demonstrate that Phi29 is strongly inhibited by high concentrations of heme, with samples derived from blood producing negative results following WGA (Ballantyne et al., Forensic Science International 166 (2007) 35-41). Because of this problem, standard WGA protocols involve purification steps to eliminate red blood cells in order to prevent decreased yields and background amplification.
Thus, there is a need for improved methods and compositions for preparing samples for isothermal WGA and array CGH (particularly methods and compositions for preparing low levels of genomic material, such as genomic material from a single cell), methods and compositions for generating consistent amplified products with low bias, and methods and compositions that can be scaled up for high throughput performance and analyses.